SF9, High Five, and BHK-21 cells were maintained in the Veterinary Microbiology and Immunology Laboratory at South China Agricultural University. SF9 and High Five cells were cultured in SIM SF Expression Medium and SIM HF Expression Medium (Sino Biological Inc., Beijing, China), respectively, under suspension conditions in a 27°C incubator shaker at 140 rpm. BHK-21 cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibco, USA) at 37°C with 5% CO₂ in a cell incubator. FMDV type O strain O/BY/CHA/2010 was propagated in BHK-21 cells using standard virology techniques and the supernatants of infected cells were clarified and stored at −80°C.

Based on the VP1, VP3, and VP0 gene sequences of the FMDV type O reference strain (GenBank: JN998085.1), a histidine (His) tag sequence encoding six histidines was introduced at the C-terminus of each protein. A dominant B-cell epitope (amino acids 200–213) was inserted into the G-H loop (positions 173–174 aa) of the VP3 protein, resulting in the gene sequences for VP1, VP3B, and VP0. Additionally, two B-cell epitopes (137–160aa and 200–213aa) and one T-cell epitope (21–40aa) from VP1 were sequentially linked together and then inserted into the VL, RGD1, and RGD2 regions of the ADDomer (Patent No. US201716088905). A His tag was added at the end of the T-cell epitope to generate the ADDomer-BBT protein gene sequence. The predicted structure of the recombinant VLP was modeled using the crystal structure files with PDB IDs 8HEE (FMDV) and 6HCR (ADDomer) and the online homology modeling website Swiss Model (http://swissmodel.expasy.org/; accessed on October 10, 2024), with structural visualization performed using PyMOL software.Based on the porcine CD154 gene sequence (GeneBank: NM_214126.1), a His tag sequence encoding six histidines was introduced at the C-terminus, resulting in the recombinant protein gene sequence for CD154. To ensure high expression levels in insect cells, the codons of the five gene sequences were optimized and synthesized by Hongxun Biotechnology Co., Ltd. (Suzhou, China). The sequences were inserted into the pFBDM vector as shown in Figures 1A–C.

The constructed recombinant transfer plasmids pFBDM-VP1-VP3_B-VP0, pFBDM-ADDomer-BBT, and pFBDM-CD154 were transformed into DH10MultiBac competent cells. The transformed cells were spread on LB agar plates containing Gen, Kan, Amp, IPTG, and X-Gal and incubated at 37°C for 48 h. White colonies were selected, and PCR identification was performed using the M13 universal primers of the pFBDM vector. The positively identified recombinant colonies were expanded and used for recombinant baculoviral plasmid extraction. The extracted recombinant baculoviral plasmids were designated as rFBDM-VP1-VP3_B-VP0, rFBDM-ADDomer-BBT, and rFBDM-CD154. Following Wu et al.'s method (26), the recombinant baculovirus plasmids were transfected into SF9 cells. After 96 h incubation at 27°C, cytopathic effects were observed, and the supernatants were collected as P1 recombinant baculoviruses, named Ac-VP1-VP3_B-VP0, Ac-ADDomer-BBT, and Ac-CD154, respectively.

To quantify the recombinant baculovirus concentration, absolute fluorescence quantitative PCR was performed. Standard pMD18T-Ac plasmids at concentrations of 1 × 10³ to 1 × 10⁸ copies/μl were used to create a standard curve. Viral DNA was extracted from Ac-VP1-VP3_B-VP0, Ac-ADDomer-BBT, and Ac-CD154 recombinant baculoviruses according to the Viral DNA Kit instructions, serving as the template for fluorescence quantitative PCR. The virus copy number was calculated using the standard curve, and the viral titer was determined using Equation 1.

The obtained recombinant baculoviruses were passaged to P3 and analyzed by Western blot. Primary antibodies included mouse monoclonal anti-His (Sino Biological Inc., Beijing, China), porcine FMDV-positive serum (Yongshun Biotechnology Co., Ltd., Guangdong, China), rabbit monoclonal anti-FMDV VP1 (Bioss Biotechnology Co., Ltd., Beijing, China), and recombinant anti-TARP/CD40L (Abcam plc, UK). Secondary antibodies were goat anti-mouse and goat anti-rabbit IgG-HRP antibodies (Beyotime Biotechnology Co., Ltd., Shanghai, China). PVDF membranes were visualized using ECL chemiluminescent solution (YaMei Biological Medicine Co., Ltd., Shanghai, China) with a gel imaging system.

Due to the lack of VP3 and VP0 monoclonal antibodies, mass spectrometry was performed on all three proteins. P3-generation SF9 cells infected with Ac-VP1-VP3_B-VP0 for 72 h were harvested, mixed with protein sample buffer, and denatured in a metal bath at 100°C for 10 min before SDS-PAGE electrophoresis. The gel was stained with BeyoBlue™ Coomassie Brilliant Blue, and the protein bands of expected sizes were excised, placed in 1.5 ml EP tubes, and sent to Biopeson Biotechnology Co., Ltd. (Shanghai, China) for protein identification via mass spectrometry.

To further verify recombinant protein expression, SF9 cells were infected with P3-generation Ac-VP1-VP3_B-VP0, Ac-ADDomer-BBT, and Ac-CD154 at an MOI of 0.1, with a negative control group (uninfected). After 72 h at 27°C, indirect immunofluorescence detection was performed using a 1:250 diluted mouse anti-His antibody as the primary antibody and a 1:1,000 diluted FITC-labeled goat anti-mouse IgG antibody as the secondary antibody.

To determine optimal harvest time and MOI for recombinant protein expression, High Five cells were used for large-scale expression, and P3 recombinant baculoviruses were inoculated at MOI values of 2.0, 5.0, and 8.0. After shaking at 27°C and 130 rpm, samples were collected at 48 h, 72 h, 96 h, and 120 h post-infection and analyzed by Western blot. Protein gray values were quantified with ImageJ software, and expression trend graphs were generated to determine the optimal MOI and harvest time in High Five cells.

Based on the optimized expression conditions in 2.6, Ac-VP1-VP3_B-VP0, Ac-ADDomer-BBT, and Ac-CD154 P3 recombinant baculoviruses were inoculated into High Five cells for large-scale protein expression. After ultrasonic disruption of collected cells, supernatants were centrifuged at 12,000 rpm for 20 min at 4°C and filtered using Millipore ultrafiltration tubes. Samples were loaded onto sucrose gradients of 70%, 50%, and 30% and centrifuged at high speed. The resulting protein bands were collected, filtered through 0.22 μm membranes, and analyzed by SDS–PAGE. The gels were stained with BeyoBlue™ Coomassie Brilliant Blue to evaluate sucrose gradient centrifugation purification results.

To investigate the morphology of the recombinant proteins, 10 μl samples were added to carbon-coated grids, stained with 2% uranyl acetate, and dried for observation under a transmission electron microscope (TEM).

A His-tagged protein at 2,000 μg/ml was serially diluted and used as a standard alongside the recombinant protein samples for Western blot analysis. ImageJ was used to analyze the gray values of the standards, and a standard curve was generated to calculate recombinant protein concentrations.

The concentrations of recombinant proteins VP1-VP3_B-VP0, ADDomer-BBT, and CD154 were adjusted with PBS buffer. The proteins were mixed with ISA 201 (SEPPIC, France) adjuvant at a 1:1 mass ratio to produce vaccines with final dosages as follows: 70 μg VP1, 50 μg VP3, and 30 μg VP0 per 2 ml dose for the FMDV VLP vaccine (VP1-VP3_B-VP0 vaccine); 50 μg of ADDomer-BBT recombinant protein per 2 ml dose for the FMDV chimeric VLP vaccine (ADDomer-BBT vaccine); and 25 μg of recombinant CD154 as a molecular adjuvant in the molecular adjuvant control groups (VP1-VP3_B-VP0 + CD154 and ADDomer-BBT + CD154). A control was prepared with PBS and ISA 201 adjuvant in a 1:1 ratio. The vaccines were assessed for appearance, formulation, and stability.

Thirty 4-week-old seronegative piglets were randomly divided into six groups (five piglets per group) as shown in Table 1. After being raised for 1 week, a primary immunization was administered by intramuscular injection. The piglets were observed daily for local and systemic adverse reactions following vaccination.

FMDV-specific antibody levels in the peripheral blood of piglets were determined using the Porcine FMDV O-type Antibody ELISA Kit, following the manufacturer's instructions (Finder Biotechnology Co., Ltd., Shenzhen, China).

Healthy BHK-21 cells were seeded into 96-well plates and cultured in a 5% CO₂ incubator at 37°C until 80% confluency. Serum samples collected on day 35 post-initial immunization were heated at 56°C for 30 min and serially diluted in DMEM containing 2% FBS. A mixture of 300 μl of the diluted serum and 300 μl of FMDV suspension containing 100 TCID50 was incubated at 37°C for 1 h. Subsequently, 100 μl of the virus-serum mixture was added to each well of the prepared BHK-21 cells, with a blank negative control included. Each dilution level was tested in quadruplicate, and the plates were incubated at 37°C in a 5% CO₂ environment for 6–8 h. After incubation, the supernatant was removed, and serum-free DMEM was added to each well, with each group set up in four replicates. Cytopathic effects (CPE) were monitored under a microscope, and the neutralizing antibody titer was calculated based on the highest serum dilution exhibiting 50% CPE.

Peripheral blood lymphocytes from piglets were isolated from blood samples collected on day 35 post-initial immunization using a lymphocyte separation kit (HaoYang Biological Products Technology Co., Ltd., Tianjin, China) according to the manufacturer's instructions. The lymphocyte concentration was adjusted to 5 × 10⁶ cells/ml. Lymphocytes were divided into positive control, negative control, and experimental groups, with each group set up in four replicates. Cells were seeded at 100 μl per well. The positive control group received 100 μl of Con A (10 μg/ml), the negative control received 100 μl of complete 1640 medium with 10% FBS, and the experimental group received 100 μl of inactivated FMDV. After 48 h of incubation at 37°C in a 5% CO₂ incubator, 10 μl of CCK-8 solution was added to each well and incubated for an additional 2–4 h. The OD450 value was measured every 30 min, and the stimulation index (SI) was calculated as SI = average OD450 (virus-stimulated or positive control group)/average OD450 (1640 complete medium group).

The levels of IFN-γ, IL-2, and IL-4 in peripheral blood from piglets were measured using ELISA kits for porcine IFN-γ (RX501082P), IL-2 (RX501069P), and IL-4 (RX501065P) in accordance with the manufacturer's instructions (Ruisen Biotechnology Co., Ltd., Fujian, China).

Data were analyzed using GraphPad Prism 9 software, applying one-way ANOVA or two-way ANOVA for statistical comparisons between groups. A probability (P) value of < 0.05 was considered statistically significant.

The structure of the VP3_B protein with a B-cell epitope inserted in the G-H loop was predicted and compared to the unmodified VP3 structure. The results showed that the inserted epitope in the central region of the G-H loop was exposed on the protein's surface (Figure 1D). Moreover, tandem antigenic epitopes were inserted into the ADDomer framework based on the insertion pattern in Figure 1E. The inserted epitopes in the VL, RGD1, and RGD2 regions are all displayed on the particle's outer surface (Figures 1F, G). The designed sequences were synthesized and inserted into the pFBDM vector (Figures 1A–C).

Recombinant baculoviral plasmids rFBDM-VP1-VP3_B-VP0, FBDM-ADDomer-BBT, and rFBDM-CD154 were transfected into adherent SF9 cells in the logarithmic growth phase. After incubation at 27°C for 72 h, cell morphology was observed. As shown in Figure 2A, compared to untransfected cells, SF9 cells transfected with recombinant baculovirus plasmids exhibited extensive detachment and cell death, with a marked increase in cell volume, nuclear swelling, and blurred cell membrane edges. The cell culture supernatant was collected, centrifuged, and used to obtain the P1 generation recombinant baculovirus, designated as Ac-VP1-VP3_B-VP0, Ac-ADDomer-BBT, and Ac-CD154.

IFA analysis of SF9 cells infected with the P3 generation of recombinant baculovirus revealed specific green fluorescence in cells infected with Ac-VP1-VP3_B-VP0, Ac-ADDomer-BBT, and Ac-CD154, while no fluorescence was detected in the negative control group, confirming the successful construction of recombinant baculovirus capable of expressing recombinant proteins (Figure 2B). Absolute quantitative PCR with established standards produced linear standard curves (Figure 2C), and the Cq values were used to calculate recombinant baculovirus copy numbers for P3 Ac-VP1-VP3_B-VP0, Ac-ADDomer-BBT, and Ac-CD154, yielding 2.55 × 10⁸, 3.972 × 10⁸, and 1.191 × 10⁸ copies/μl, respectively. Then, the viral titers were calculated according to the formula, resulting in titers of 4.811 × 10⁹ pfu/ml, 7.494 × 10⁹pfu/ml, and 1.767 × 10⁹ pfu/ml.

Using a mouse-derived anti-His tag antibody and FMDV-positive serum as primary antibodies, the recombinant VP1-VP3_B-VP0 protein expression was identified by Western blot analysis, with specific bands observed at ~24 kDa, 25 kDa, and 30 kDa (Figure 3A). To further confirm VP1-VP3_B-VP0 protein expression, mass spectrometry analysis was performed using the original files processed with MasQuant 2.0.1.0 based on the supplementary parameters in Table 2, verifying the presence of VP1, VP3_B, and VP0. The mass spectrometry results are shown in Supplementary Figure S1. For the ADDomer-BBT recombinant protein, Western blot analysis using mouse-derived anti-His and rabbit-derived anti-FMDV VP1 monoclonal antibodies as primary antibodies showed a specific band at 71 kDa (Figure 3B). For the recombinant CD154 protein, Western blot analysis using mouse-derived anti-His and anti-TARP/CD40L antibodies as primary antibodies revealed a specific band at 30 kDa (Figure 3C).

P3 generation recombinant baculovirus was inoculated into suspension High Five cells to optimize the inoculum amount and protein harvest time. Figure 3D shows the results. For practical production cost considerations, an MOI of 5.0 was selected as the optimal dose for Ac-CD154 recombinant baculovirus, with CD154 protein harvested at 72 h post-inoculation. For Ac-ADDomer-BBT, the optimal MOI was 5.0, with ADDomer-BBT protein harvested at 96 h post-inoculation. Similarly, an MOI of 5.0 was used for Ac-VP1-VP3_B-VP0, with protein harvested at 72 h.

Purified recombinant proteins obtained under optimal expression conditions were separated by 30%, 50%, and 70% sucrose gradient centrifugation. Protein bands (“protein rings”) were observed in the interlayers of 30%, 30–50%, and 50–70% sucrose (Figure 4A). SDS-PAGE analysis of these layers revealed specific protein bands with minimal non-specific bands: VP1-VP3_B-VP0 at 24, 25, and 30 kDa; ADDomer-BBT at 71 kD; and CD154 at 30 kDa (Figure 4B). Using 2% uranyl acetate staining, TEM showed that the VLP assembled by VP1-VP3_B-VP0 was 25–30 nm in diameter, while the VLP assembled by ADDomer-BBT was 30–40 nm (Figure 4C). Recombinant protein concentrations were determined by measuring the gradient of His-tagged standards: VP0, VP1, and VP3_B in VP1-VP3_B-VP0 were 51.01 μg/ml, 117.37 μg/ml, and 83.72 μg/ml, respectively; ADDomer-BBT and CD154 concentrations were 239.04 μg/ml and 371.74 μg/ml (Figure 4D).

The FMDV VLP vaccine formulated with ISA 201 adjuvant formed a white water-in-oil-in-water (W/O/W) emulsion. Upon centrifugation at 3,000 rpm, 4°C for 15 min, no phase separation occurred, indicating the vaccine's stability. Thirty FMDV antigen- and antibody-negative piglets were immunized according to the strategy in Figure 5A. At 35 days post-inoculation, all piglets survived with normal behavior, appetite, and stable body temperature (Figure 5B). No adverse reactions were observed at the injection site.

Peripheral blood was collected every seven days after the primary immunization to detect serum FMDV-specific antibody levels. At 35 days post-primary immunization, specific antibody levels in the ADDomer-BBT+CD154 vaccine group were higher than the commercial vaccine group, with no significant difference (P > 0.05; Figure 6A). The VP1-VP3_B-VP0 + CD154 group showed antibody levels comparable to the commercial group (P > 0.05). Antibody levels in the ADDomer-BBT group were slightly higher than the VP1-VP3_B-VP0 group, without significant difference (P > 0.05). Specific antibody levels were significantly higher in the VP1-VP3_B-VP0 + CD154 group than in the VP1-VP3_B-VP0 group (P < 0.05), and in the ADDomer-BBT + CD154 group than in the ADDomer-BBT group (P < 0.05), indicating that CD154 can enhance the humoral immune response of FMDV VLP-induced immunity in piglets.

To assess protective efficacy, FMDV neutralizing antibody levels in piglet serum were measured 35 days post-primary immunization. As shown in Figure 6B, all vaccine groups showed high neutralizing antibody titers, comparable to the commercial vaccine (P > 0.05), except for the PBS group. Neutralizing antibody levels in the VP1-VP3_B-VP0 + CD154 and ADDomer-BBT + CD154 groups were significantly higher than in the VP1-VP3_B-VP0 and ADDomer-BBT groups (P < 0.05), confirming the ability of CD154 to enhance humoral immune effects.

To evaluate immune response levels post-vaccination, lymphocyte proliferation in piglet peripheral blood was measured using the CCK-8 method. Results indicated that all vaccine groups showed a significantly higher stimulation index (SI) than the PBS group (P < 0.01). The ADDomer-BBT group had a slightly higher SI than the VP1-VP3_B-VP0 group, with no significant difference (P > 0.05). VLP vaccine groups containing CD154 showed higher SI than those without CD154 (P < 0.05). These findings suggest that vaccines developed in this study significantly enhance the immune response against FMDV in piglets, strengthening their resistance to viral infection (Figure 6C).

To investigate the host cellular immune mechanisms following vaccination, indirect ELISA kits were used to measure IL-2, IFN-γ, and IL-4 levels in the peripheral blood of piglets at 35 days post-primary immunization. These cytokine levels were assessed to determine the Th1- and Th2-type immune responses induced by the vaccines. The results (Figures 7A, C) indicated that IL-2 and IFN-γ levels in all vaccinated groups were significantly higher than those in the PBS control group (P < 0.05). Furthermore, the IL-2 and IFN-γ levels in the VP1-VP3_B-VP0 + CD154 group were significantly higher than those in the VP1-VP3_B-VP0 group (P < 0.05). Similarly, the ADDomer-BBT + CD154 group exhibited IL-2 and IFN-γ levels that were significantly elevated compared to those in the ADDomer-BBT group. These findings suggest that the VP1-VP3_B-VP0 and ADDomer-BBT recombinant proteins effectively induce IL-2 and IFN-γ secretion, initiating a Th1-type cellular immune response, while CD154, as an adjuvant, enhances the vaccine's ability to stimulate IL-2 and IFN-γ production.

For IL-4 levels across all vaccinated groups, each group displayed significantly higher IL-4 secretion compared to the PBS control group (P < 0.01), and the addition of CD154 further enhanced IL-4 levels (Figure 7B). This result indicates that each VLP vaccine promotes IL-4 secretion, thereby inducing a Th2-type cellular immune response.